31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Tumor hypoxia is associated with resistance to PD-1 blockade in squamous cell carcinoma of the head and neck

The majority of patients with recurrent/metastatic squamous cell carcinoma of the head and neck (HNSCC) (R/M) do not benefit from anti-PD-1 therapy. Hypoxia induced immunosuppression may be a barrier to immunotherapy. Therefore, we examined the metabolic effect of anti-PD-1 therapy in a murine MEER HNSCC model as well as intratumoral hypoxia in R/M patients. In order to characterize the tumor microenvironment in PD-1 resistance, a MEER cell line was created from the parental line that are completely resistant to anti-PD-1. These cell lines were then metabolically profiled using seahorse technology and injected into C57/BL6 mice. After tumor growth, mice were pulsed with pimonidazole and immunofluorescent imaging was performed to analyze hypoxia and T cell infiltration. To validate the preclinical results, we analyzed tissues from R/M patients (n=36) treated with anti-PD-1 mAb, via immunofluorescent imaging for number of CD8+ T cells (CD8), Tregs and the percent area (CAIX) and mean intensity (I) of carbonic anhydrase IX in tumor. We analyzed disease control rate (DCR), progression free survival (PFS), and overall survival (OS) using proportional odds and proportional hazards (Cox) regression. We found that anti-PD-1 resistant MEER has significantly higher oxidative metabolism, while there was no difference in glycolytic metabolism. Intratumoral hypoxia was significantly increased and CD8+ T cells decreased in anti-PD-1 resistant tumors compared with parental tumors in the same mouse. In R/M patients, lower tumor hypoxia by CAIX/I was significantly associated with DCR (p=0.007), PFS, and OS, and independently associated with response (p=0.028) and PFS (p=0.04) in a multivariate model including other significant immune factors. During PD-1 resistance, tumor cells developed increased oxidative metabolism leading to increased intratumoral hypoxia and a decrease in CD8+ T cells. Lower tumor hypoxia was independently associated with increased efficacy of anti-PD-1 therapy in patients with R/M HNSCC. To our knowledge this is the first analysis of the effect of hypoxia in this patient population and highlights its importance not only as a predictive biomarker but also as a potential target for therapeutic intervention

Early TCR Signaling Induces Rapid Aerobic Glycolysis Enabling Distinct Acute T Cell Effector Functions

To fulfill bioenergetic demands of activation, T cells perform aerobic glycolysis, a process common to highly proliferative cells in which glucose is fermented into lactate rather than oxidized in mitochondria. However, the signaling events that initiate aerobic glycolysis in T cells remain unclear. We show T cell activation rapidly induces glycolysis independent of transcription, translation, CD28, and Akt and not involving increased glucose uptake or activity of glycolytic enzymes. Rather, TCR signaling promotes activation of pyruvate dehydrogenase kinase 1 (PDHK1), inhibiting mitochondrial import of pyruvate and facilitating breakdown into lactate. Inhibition of PDHK1 reveals this switch is required acutely for cytokine synthesis but dispensable for cytotoxicity. Functionally, cytokine synthesis is modulated via lactate dehydrogenase, which represses cytokine mRNA translation when aerobic glycolysis is disengaged. Our data provide mechanistic insight to metabolic contribution to effector T cell function and suggest that T cell function may be finely tuned through modulation of glycolytic activity. Menk et al. show rapid induction of aerobic glycolysis after activation of effector T cells that is required for acute cytokine production. These data provide mechanistic insight into the regulation of T cell function through nutrient availability

Checkpoint molecules coordinately restrain hyperactivated effector T cells in the tumor microenvironment

The immune checkpoint blockade (ICB) immunotherapy has prolonged overall survival for cancer patients but the response rates are low. The resistance to ICB is likely due to compensatory upregulation of additional immune inhibitory molecules. In this study, we first systematically examined Tim-3 expression in immune cells in mouse tumors and found that Tim-3 was specifically up-regulated in a large number of Treg, conventional CD4+, CD8+ T cells, dendritic cell 1 (DC1), and macrophage 1 (M1) in the tumor microenvironment (TME). Interestingly, Tim-3+ T cells in the TME were phenotypically effector but not “exhausted” T cells because Tim-3+ PD-1+ CD8+ T cells had a higher number of mitochondria, greater levels of glycolysis, and higher tumor-specific cytolytic activities compared to Tim-3− PD-1− CD8+ T cells. The combination treatment with Tim-3 and PD-1 mAbs resulted in a synergistic antitumor activity but also increased the expression of Lag-3 and GITR in TIL, demonstrating cross-regulation between multiple checkpoint molecules. Furthermore, we found that the antitumor efficacy with triple combination of Tim-3, PD-1, and Lag3 mAbs was much greater than any two antibodies. Mechanistically, we demonstrated that simultaneous targeting of Tim-3, PD-1, and Lag-3 cooperatively increased the levels of granzyme B and tumor-specific cytolytic activities of CD8+ TIL. Our data indicate that multiple checkpoint molecules are coordinately upregulated to inhibit the function of hyperactivated T cells in the TME and requirement for the simultaneous blockade of PD-1, Tim-3 and Lag3 for cancer treatment

Early TCR Signaling Induces Rapid Aerobic Glycolysis Enabling Distinct Acute T Cell Effector Functions

Summary: To fulfill bioenergetic demands of activation, T cells perform aerobic glycolysis, a process common to highly proliferative cells in which glucose is fermented into lactate rather than oxidized in mitochondria. However, the signaling events that initiate aerobic glycolysis in T cells remain unclear. We show T cell activation rapidly induces glycolysis independent of transcription, translation, CD28, and Akt and not involving increased glucose uptake or activity of glycolytic enzymes. Rather, TCR signaling promotes activation of pyruvate dehydrogenase kinase 1 (PDHK1), inhibiting mitochondrial import of pyruvate and facilitating breakdown into lactate. Inhibition of PDHK1 reveals this switch is required acutely for cytokine synthesis but dispensable for cytotoxicity. Functionally, cytokine synthesis is modulated via lactate dehydrogenase, which represses cytokine mRNA translation when aerobic glycolysis is disengaged. Our data provide mechanistic insight to metabolic contribution to effector T cell function and suggest that T cell function may be finely tuned through modulation of glycolytic activity

Metabolic reprogramming via an engineered PGC-1α improves human chimeric antigen receptor T-cell therapy against solid tumors

Background Cellular immunotherapies for cancer represent a means by which a patient’s immune system can be augmented with high numbers of tumor-specific T cells. Chimeric antigen receptor (CAR) therapy involves genetic engineering to ‘redirect’ peripheral T cells to tumor targets, showing remarkable potency in blood cancers. However, due to several resistance mechanisms, CAR-T cell therapies remain ineffective in solid tumors. We and others have shown the tumor microenvironment harbors a distinct metabolic landscape that produces a barrier to immune cell function. Further, altered differentiation of T cells within tumors induces defects in mitochondrial biogenesis, resulting in severe cell-intrinsic metabolic deficiencies. While we and others have shown murine T cell receptor (TCR)-transgenic cells can be improved through enhanced mitochondrial biogenesis, we sought to determine whether human CAR-T cells could be enabled through a metabolic reprogramming approach.Materials and methods Anti-EGFR CAR-T cells were infused in NSG mice which bore A549 tumors. The tumor infiltrating lymphocytes were analyzed for exhaustion and metabolic deficiencies. Lentiviruses carrying PPAR-gamma coactivator 1α (PGC-1α), PGC-1αS571A and NT-PGC-1α constructs were used to co-transduce T cells with anti-EGFR CAR lentiviruses. We performed metabolic analysis via flow cytometry and Seahorse analysis in vitro as well as RNA sequencing. Finally, we treated therapeutically A549-carrying NSG mice with either PGC-1α or NT-PGC-1α anti-EGFR CAR-T cells. We also analyzed the differences in the tumor-infiltrating CAR-T cells when PGC-1α is co-expressed.Results Here, in this study, we show that an inhibition resistant, engineered version of PGC-1α, can metabolically reprogram human CAR-T cells. Transcriptomic profiling of PGC-1α-transduced CAR-T cells showed this approach effectively induced mitochondrial biogenesis, but also upregulated programs associated with effector functions. Treatment of immunodeficient animals bearing human solid tumors with these cells resulted in substantially improved in vivo efficacy. In contrast, a truncated version of PGC-1α, NT-PGC-1α, did not improve the in vivo outcomes.Conclusions Our data further support a role for metabolic reprogramming in immunomodulatory treatments and highlight the utility of genes like PGC-1α as attractive candidates to include in cargo along with chimeric receptors or TCRs for cell therapy of solid tumors

Lymphocyte Activation Gene-3 Maintains Mitochondrial and Metabolic Quiescence in Naive CD4+ T Cells

Summary: Lymphocyte activation gene-3 (LAG-3) is an inhibitory receptor expressed by CD4+ T cells and tempers their homeostatic expansion. Because CD4+ T cell proliferation is tightly coupled to bioenergetics, we investigate the role of LAG-3 in modulating naive CD4+ T cell metabolism. LAG-3 deficiency enhances the metabolic profile of naive CD4+ T cells by elevating levels of mitochondrial biogenesis. In vivo, LAG-3 blockade partially restores expansion and the metabolic phenotype of wild-type CD4+ T cells to levels of Lag3−/− CD4+ T cells, solidifying that LAG-3 controls these processes. Lag3−/− CD4+ T cells also demonstrate greater signal transducer and activator of transcription 5 (STAT5) activation, enabling resistance to interleukin-7 (IL-7) deprivation. These results implicate this pathway as a target of LAG-3-mediated inhibition. Additionally, enhancement of STAT5 activation, as a result of LAG-3 deficiency, contributes to greater activation potential in these cells. These results identify an additional mode of regulation elicited by LAG-3 in controlling CD4+ T cell responses. : Previte et al. show that LAG-3 expression regulates the metabolic profile of naive CD4+ T cells during homeostatic expansion. They observed that Lag3-deficient CD4+ T cells are resistant to Interleukin-7 deprivation due to enhanced STAT5 activation. Increased STAT5 signaling also mediated greater activation potential in these T cells following stimulation. Keywords: LAG-3, CD4+ T cell, metabolism, mitochondria, STAT